Thrombopoietin proteins with improved properties

ABSTRACT

The invention concerns human thrombopoietin and in particular modified forms of thrombopoietin (TPO) with improved properties. The improved proteins contain amino acid substitutions at specific positions within the TPO molecule. The invention provides modified TPO molecules, preferably fusion proteins comprising immunoglobulin constant regions and modified human TPO, with improved biological activity concomitant with reduced immunogenic potential in the protein. The improved proteins are intended for therapeutic use in the treatment of diseases in humans.

This application is the National Stage of International Application No.PCT/EP2004/006887, filed on Jun. 25, 2004.

FIELD

The invention concerns human thrombopoietin and in particular modifiedforms of thrombopoietin (TPO) with improved properties. The improvedproteins contain amino acid substitutions at specific positions withinthe TPO molecule. The invention provides modified TPO molecules,preferably fusion proteins comprising immunoglobulin constant regionsand modified human TPO, with improved biological activity concomitantwith reduced immunogenic potential in the protein. The improved proteinsare intended for therapeutic use in the treatment of diseases in humans.

BACKGROUND

Thrombopoietin (TPO) is a glycoprotein hormone involved regulation ofplatelet production. TPO promotes both the proliferation ofmegakaryocyte progenitors in the bone marrow and their maturation intoplatelet-producing megakaryocytes.

TPO has significant therapeutic value in the treatment of patients withreduced platelet count. In particular patients with many types of cancersuffer thrombocytopenias on account of myelosuppressive chemotherapy.Platelet transfusion has historically been the mainstay by which suchpatients have been supported. The availability of purified TPO fromrecombinant sources could enhance the options available for aggressivechemotherapy regimens and other patients at risk of bleedingcomplications due to their thrombocytopenia [Prow, D. & Vadhan-Raj, S.(1998) Oncology 12: 1597-1608].

At least two forms of recombinant human TPO have been developed forclinical trails. A truncated version comprising only the N-terminal 163amino acids conjugated with polyethylene glycol is referred to aspegylated recombinant human megakaryocyte growth and development factor(PEG-rHuMDGF). A full length and glycosylated molecule is referred to asrecombinant human thrombopoietin (rhTPO).

Both forms of TPO have been evaluated in Phase I/II trials, where theywere given to cancer patients before receiving chemotherapy in order toboost platelet counts. The results of these trials have been reported[Basser R. L. et al (1996) Lancet; 348: 1279-1281; Basser R. L. et al(1997) Blood; 89: 3118-3128. Erratum in 1997; 90: 2513; Fannuchi M. etal, (1997), New England Journal of Medicine; 336: 404-409; Vadhan-Raj S.et al. (1997) Ann. Intern. Med; 126: 673-681 and Vadhan-Raj S. (1998)Semin. Hematol; 35: 261-268].

Both forms of TPO have been found to be immunogenic in a smallproportion of subjects, and neutralising antibodies have also beendemonstrated to both molecules [Hardy L, et al (1997) The Toxicologist;36: 277; Li J, et al (2001) Blood; 98: 3241-3248; Koren E. (2002) DevBiol (Basel); 109: 87-95; Basser R. L. et al (2002), Blood; 99:2599-2602 and Koren E. (2002) Current Pharmaceutical Biotechnology;3:349-360].

Clinical trials of PEG-rHuMDGF were abandoned in 1998 as neutralisingantibodies could bind to endogenous TPO causing some of the patients andnormal volunteers involved in the trials to become platelet transfusiondependent for several years. [Neumann T. A & Foote M. (2000) CytokinesCell Mol Ther.: 6; 47-56].

Clearly for these subjects, an immune response has been mounted to thetherapeutic TPO despite the fact that TPO is normally present in thecirculation. The pivotal feature leading to the induction of an immuneresponse is the presence within the protein of peptides that canstimulate the activity of T-cells via presentation on MHC class IImolecules. Such peptide sequences are “T-cell epitopes” and are commonlydefined as any amino acid residue sequence with the ability to bind toMHC Class II molecules. Implicitly, a “T-cell epitope” means an epitopewhich when bound to MHC molecules can be recognised by a T-cell receptor(TCR), and which can, at least in principle, cause the activation ofthese T-cells by engaging a TCR to promote a T-cell response. Patientswho develop antibodies to TPO possess T cells that are capable ofrecognising peptide fragments of TPO bound to MHC class II molecules intheir T cell repertoire.

To date no form of TPO has received regulatory approval as a therapeuticcompound. From the foregoing there is clearly a continued need for TPOanalogues with enhanced properties. There is a particular need forenhancement of the in vivo characteristics when administered to thehuman subject. In this regard, it is highly desired to provide TPO withreduced or absent potential to induce an immune response and enhancedbiological potency in the human subject.

Others have provided TPO molecules and analogues [U.S. Pat. Nos.5,989,538; U.S. Pat. No. 6,083,913; 5,879,673] including chemicallymodified and truncated forms [U.S. Pat. No. 5,989,538] and TPO fusionproteins [U.S. Pat. No. 6,066,318].

Koren et al [US Patent Application 20030077756] have identified peptidesequences in the C-terminal domain of human TPO that are able tointeract with anti-TPO antibodies.

None of these teachings recognise the importance of T cell epitopes tothe immunogenic properties of the protein nor have been conceived todirectly influence said properties in a specific and controlled wayaccording to the scheme of the present invention.

WO 03/104263 describes methods for the identification of CD4+ T-cellepitopes in cytokines including TPO. According to this method an epitopeat residues 154-171 was defined. However, substitutions leading to adesired altered immunogenic response were suggested at residues outsideof the epitope at residues 138, 139 and 140.

The co-owned application WO 02/068469 describes the results of ananalysis of the entire TPO sequence for the presence of potential MHCclass II binding ligands. The analysis therein is conducted using acomputer simulation of the peptide MHC binding interaction. WO 02/068469also provides multiple amino acid substitutions for achieving thedisruption of the said potential epitope sequences.

The present invention is concerned also with TPO molecules in whichamino acid substitution and or combinations of substitution have beenconducted. In the present case, the molecules of the invention arefusion proteins comprising a human immunoglobulin constant region moietylinked with a human TPO mutein. Linkage to the immunoglobulin constantregion domain causes the protein to become dimeric and these moleculesadditionally show increased potency.

This structure together with substitutions and combinations ofsubstitutions in the TPO component confer the property of enhancing thebiological activity of the molecule and also achieve a reducedimmunogenic profile for the protein.

The general category of “human Fc fusion proteins” of which the presentmolecules are examples have been described previously [U.S. Pat. Nos.5,541,087; 5,726,044 Lo et al (1998), Protein Engineering 11:495-500].

SUMMARY OF THE INVENTION

The invention provides human thrombopoietin molecules containing aminoacid substitutions. The amino acid substitutions confer improvedproperties to the protein. The improved properties concern the specificbiological activity of the protein and also the immunogenic propertiesof the protein.

The molecules of the invention are fusion proteins comprising a humanimmunoglobulin heavy chain constant region moiety linked with a humanTPO mutein derived from wild-type truncated TPO (1-174).

The TPO proteins of the invention preferably are expressed in mammaliancell-lines as a C-terminal fusion partner, linked to the Fc unit ofhuman IgG₄, wherein the Fc portion may include a hinge region.

The TPO sequence is fused preferably to the C-terminus of a hingemodified/C_(H)2/C_(H)3 Fc region of human IgG₄ via a 15 amino acidflexible linker between the C-terminus of the C_(H)3 and the N-terminusof TPO₍₁₋₁₇₄₎. The expressed fusion proteins are dimeric and have astoichiometry of (hinge-C_(H)2—C_(H)3-linker-TPO₍₁₋₁₇₄₎)₂.

The molecules of the invention have new properties. Such molecules maycause benefit for a patient with thrombocytopenia.

The molecules of the invention are characterised by the proteinsequences defined herein as M1 to M67, F-M1 to F-M67, and F-L-M1 toF-L-M67, respectively, wherein M1 to M67 represent the protein sequencesof differently modified human TPO in its truncated form (1-174), F-M1 toF-M67 represent the respective fusion proteins with the Fc portion ofhuman IgG4 or optionally another human IgG form, and F-L-M1 to F-L-M67represent the respective fusion proteins comprising a linker moleculebetween the Fc sequence and the TPO protein sequence, wherein saidlinker molecule is preferably a linker peptide comprising 4-20 aminoacid residues.

The molecules of the invention are further characterised their relativeactivity in a proliferation assay of between 0.1 and 6.3.

A most preferred molecule of the invention is characterised by theprotein sequence M67 or F-M67 or F-L-M67 or F1-L1-M67, wherein F is a Fcportion, preferably deriving from human IgG4 and including a modifiedhinge, and L is a peptide linker of 15 amino acid residues and F1 and L1are specific sequences according to Table A3 and A4. These molecules arefurther characterised by a relative activity of around 0.4 in aproliferation assay.

A further preferred molecule of the invention is characterised by theprotein sequences M1, F-M1, F-L-M1, and preferably F1-L1-M1 and isfurther characterised by a relative activity of around 1.0 in aproliferation assay.

A further preferred molecule of the invention is characterised by theprotein sequences M66, F-M66, F-L-M66, and preferably F1-L1-M66 and isfurther characterised by a relative activity of around 0.2 in aproliferation assay.

The most preferred molecules of the invention are characterised yetfurther still by comprising sequences demonstrated to show reducedimmunogenicity in human cells. In particular reduced immunogenicity asmeasured using a “T-cell assay” or a “time course assay” as definedherein.

The present invention provides for modified forms of TPO proteins,preferably immunoglobulin fusion proteins having the biological activityof human TPO, that are expected to display enhanced properties in vivo.The present invention discloses the major regions of the TPO primarysequence that are immunogenic in man and provides modification to thesequences to eliminate or reduce the immunogenic effectiveness of thesesites.

In one embodiment, synthetic peptides comprising the immunogenic regionscan be provided in pharmaceutical composition for the purpose ofpromoting a tolerogenic response to the whole molecule.

In a further embodiment, the modified TPO molecules of the presentinvention can be used in pharmaceutical compositions.

In summary the invention is concerned with the following issues:

-   -   A modified TPO molecule (M) in a truncated (1-174) form having        essentially the same biological specificity and activity of        human TPO when used in vivo containing one or more amino acid        substitutions, wherein said modified TPO molecule is        substantially non-immunogenic or less immunogenic than the        parental non-modified human TPO and said amino acid        substitutions cause a reduction or an elimination of one or more        of T-cell epitopes within the wild-type TPO sequence which act        in the parental non-modified molecule as MHC class II binding        ligands and stimulate T-cells.    -   A modified TPO molecule as specified containing one or more of        the amino acid substitutions containing at least the amino acid        substitutions M55K, A60R and V161A within the TPO sequence.    -   A modified human TPO molecule having the formula/structure (M)

SPAPPACDLRVLSKLLRDSHVLHSRLSQCPEVHPLPTPVLLPAVDFSLGX¹X²KTQX³EEX⁴KX⁵X⁶DX⁷LGAX⁸TX⁹LX¹⁰X¹¹TVMAARGQLGPTCLSSLLGQLSGQVRLLLGALQSLLGTQLPPQGRTTAHKDPNAIFLSFQHLLRGKVRFLMLVGGSTLCVRRAPPTTAX¹²X¹³SRTSLVLTLNEL (SEQ ID NO: 1),wherein

-   -   X¹ is A, E;    -   X² is S, W;    -   X³ is A or T or K, S or M;    -   X⁴ is A, T;    -   X⁵ is R, A;    -   X⁶ is A or T or Q;    -   X⁷ is A or T or I;    -   X⁸ is A or T or V;    -   X⁹ is A or T or S or L;    -   X¹⁰ is A or L;    -   X¹¹ is A or S or E;    -   X¹² is N or A or T or R or E or D or G or H or P or K or Q or V;    -   X¹³ is A or P,    -   and whereby simultaneously X¹=E, X²=W, X³=M, X⁴=T, X⁵=A, X⁶=Q,        X⁷=I, X⁸=V, X⁹=L, X¹⁰=L, X¹¹=E, X¹²=V and X¹³=P are excluded,        said meanings representing the native human TPO.    -   A modified TPO molecule (M) as specified having a protein        sequence selected from the group consisting of M1 to M67,        wherein M1-M67 are specified in Table A1.    -   A modified TPO molecule (M) as specified having a protein        sequence of M1, M67 or M68 as specified in Table A1.    -   A fusion protein of the structure        F-(L)n-M    -    comprising a modified human TPO molecule (M) as specified,        fused directly (n=0) or indirectly (n=1) via a linker        molecule (L) to a human immunoglobulin heavy constant region        domain (F).    -   A fusion protein as specified, wherein F is an Fc domain,        optionally comprising a hinge region, wherein this hinge region        may be modified.    -   A fusion protein as specified wherein the C-terminus of the        human immunoglobulin heavy constant region domain (Fc domain) is        linked directly or indirectly to the N-terminus of the modified        TPO.    -   A dimeric fusion protein comprising two monomeric fusion protein        chains as specified.    -   A fusion protein as specified, wherein said TPO portion contains        one or more of the amino acid substitutions M55K, A60R and V161A        within the TPO domain.    -   A fusion protein as specified, wherein said TPO portion has the        formula/structure (M):

SPAPPACDLRVLSKLLRDSHVLHSRLSQCPEVHPLPTPVLLPAVDFSLGX¹X²KTQX³EEX⁴KX⁵X⁶DX⁷LGAX⁸TX⁹LX¹⁰X¹¹GVMAARGQLGPTCLSSLLGQLSGQVRLLLGALQSLLGTQLPPQGRTTAHKDPNAIFLSFQHLLRGKVRFLMLVGGSTLCVRRAPPTTAX¹²X¹³SRTSLVLTLNEL (SEQ ID NO: 1),wherein

-   -   X¹ is A, E;    -   X² is S, W;

X³ is A or T or K, S or M;

-   -   X⁴ is A, T;    -   X⁵ is R, A;    -   X⁶ is A or T or Q;    -   X⁷ is A or T or I;    -   X⁸ is A or T or V;    -   X⁹ is A or T or S or L;    -   X¹⁰ is A or L;    -   X¹¹ is A or S or E;    -   X¹² is N or A or T or R or E or D or G or H or P or K or Q or V;    -   X¹³ is A or P,    -   and whereby simultaneously X¹=E, X²=W, X³=M, X⁴=T, X⁵=A, X⁶=Q,        X⁷=I, X⁸=V, X⁹=L, X¹⁰=L, X¹¹=E, X¹²=V and X¹³=P are excluded.    -   A fusion protein as specified in Table A5 or A6, wherein said        TPO portion has a protein sequence selected from the group M1 to        M67, wherein M1-M67 are specified in Table A1.    -   A fusion protein as specified in Table A5 or A6, wherein F has        the sequence F1 as specified in Table A3.    -   A fusion protein as specified in Table A5 or A6, wherein L has        the sequence L1 as specified in Table A4.    -   A fusion protein as specified selected from the group consisting        of a member of Table A7.    -   A fusion protein selected from the group comsisting of        -   F-M1, F-L-M1, F1-L1-M1;        -   F-M66, F-L-M66, F1-L1-M66, and        -   F-M67, F-L-M67, F1-L1-M67,    -    wherein F is an immunoglobulin heavy chain constant region, F1        is the immunoglobulin heavy chain constant region of Table A3, L        is a linker molecule, and L1 is the linker peptide of Table A4.    -   A peptide molecule selected from the group consisting of        -   GEWKTQMEETKAQDILGAVTLLLEGVM (SEQ ID NO: 2),        -   PTTAVPSRTSLVLTL (SEQ ID NO: 3);    -    or a sequence track consisting of at least 9 consecutive amino        acid residues of any of said peptide molecules having a        potential MHC class II binding activity and created from the        primary sequence of non-modified human TPO in its truncated form        (1-174), whereby said peptide molecule or sequence track has a        stimulation index of >1.8 in a biological assay of cellular        proliferation and said index is taken as the value of cellular        proliferation scored following stimulation by a peptide and        divided by the value of cellular proliferation scored in control        cells not in receipt peptide and wherein cellular proliferation        is measured by any suitable means.    -   Use of said peptide molecule for the manufacture of a vaccine in        order to reduce immunogenicity to TPO in a patient    -   A modified peptide molecule deriving from any peptide molecule        as specified having a reduced or absent potential MHC class II        binding activity expressed by a stimulation index of less than        2, whereby said index is taken as the value of cellular        proliferation scored following stimulation by a peptide and        divided by the value of cellular proliferation scored in control        cells not in receipt peptide and wherein cellular proliferation        is measured by any suitable means.    -   Use of said modified peptide molecule for the manufacture of a        modified TPO molecule or a fusion protein comprising an Fc        portion of an immunoglobulin and said modified TPO.    -   A modified TPO molecule having the biological activity of human        thrombopoietin and comprising a human Fc domain and containing        at least the amino acid substitutions A60R and V161A within the        thrombopoietin domain and being further characterised by        exhibiting a relative activity of around 1.0;    -   A modified TPO molecule having the biological activity of human        thrombopoietin and comprising a human Fc domain and containing        at least the amino acid substitutions M55K, A60R and V161A        within the thrombopoietin domain and being further characterised        by exhibiting a relative activity of around 0.4;

The mutant proteins of the present invention are readily made usingrecombinant DNA techniques well known in the art and the inventionprovides methods for the recombinant production of such molecules.

In as far as this invention relates to modified TPO, compositionscontaining such modified TPO proteins or fragments of modified TPOproteins and related compositions should be considered within the scopeof the invention. In another aspect, the present invention relates tonucleic acids encoding modified TPO entities. In a further aspect thepresent invention relates to methods for therapeutic treatment of humansusing the modified TPO proteins.

DETAILED DESCRIPTION OF THE INVENTION

In nature, the mature TPO protein is single polypeptide of 332 aminoacids The amino acid sequence of TPO (depicted as single-letter code) isas follows (M68):

SPAPPACDLRVLSKLLRDSHVLHSRLSQCPEVHPLPTPVLLPAVDFSLGEWKTQMEETKAQDILGAVTLLLEGVMAARGQLGPTCLSSLLGQLSGQVRLLLGALQSLLGTQLPPQGRTTAHKDPNAIFLSFQHLLRGKVRFLMLVGGSTLCVRRAPPTTAVPSRTSLVLTLNELPNRTSGLLETNFTASARTTGSGLLKWQQGFRAKIPGLLNQTSRSLDQIPGYLNRIHELLNGTRGLFPGPSRRTLGAPDISSGTSDTGSLPPNLQPGYSPSPTHPPTGQYTLFPLPPTLPTPVVQLHPLLPDPSAPTPTPTSPLLNTSYTHSQNLSQEG (SEQ ID NO: 4).

The mature protein comprises distinct regions with the N-terminal domainhighly conserved between mouse and man and significant homology witherythropoietin and interferon-alpha and interferon-beta [de Sauvage, F.J. et al (1994) Nature 369: 533-538; Chang, M. et al (1995) J. Biol.Chem. 270: 511-514. The C-terminal domain has several sites for N-linkedglycosylation. The N-terminal domain is sufficient for thethrombopoietic effect of the molecule whereas the C-terminal region islikely important in maintaining the circulating half-life in vivo[Foster, D. et al (1996) Stem Cells 14: 102-107].

The term “TPO” is used herein to denote human thrombopoietin. In someinstances the term is also used more broadly herein to include fusionproteins (see below) comprising a TPO moiety and or more especially aTPO mutein.

The term “mutein” is used herein to denote a TPO protein engineered tocontain one or more amino acid substitutions differing from the abovenative sequence.

In addition, the TPO muteins of the invention each represent a truncatedversion of the native sequence and comprise residues only residues 1-174of the above sequence thereby encompassing the complete N-terminaldomain of the native protein.

“TPO muteins” and “TPO fusion proteins” according to the invention referto proteins comprising a TPO domain of 174 residues.

Other TPO muteins and TPO fusion proteins comprising more or less than174 residues of TPO sequence may be contemplated and fall under thescope of the present. Thus TPO fusion proteins comprising residues 1-164or 1-165 or 1-166 or 1-167 or 1-168 or 1-169 or 1-170 or 1-171 or 1-172or 1-173 can be contemplated and may be expected to have propertiesequivalent to the preferred molecules of the invention.

The term “peptide” as used herein, is a compound that includes two ormore amino acids. The amino acids are linked together by a peptide bond.

A peptide bond is the sole covalent linkage between amino acids in thelinear backbone structure of all peptides, polypeptides or proteins. Thepeptide bond is a covalent bond, planar in structure and chemicallyconstitutes a substituted amide. An “amide” is any of a group of organiccompounds containing the grouping —CONH—.

There are 20 different naturally occurring amino acids involved in thebiological production of peptides, and any number of them may be linkedin any order to form a peptide chain or ring. The naturally occurringamino acids employed in the biological production of peptides all havethe L-configuration. Synthetic peptides can be prepared employingconventional synthetic methods, utilizing L-amino acids, D-amino acids,or various combinations of amino acids of the two differentconfigurations. Some peptides contain only a few amino acid units. Shortpeptides, e.g., having less than ten amino acid units, are sometimesreferred to as “oligopeptides”. Other peptides contain a large number ofamino acid residues, e.g. up to 100 or more, and are referred to as“polypeptides”. By convention, a “polypeptide” may be considered as anypeptide chain containing three or more amino acids, whereas a“oligopeptide” is usually considered as a particular type of “short”polypeptide. Thus, as used herein, it is understood that any referenceto a “polypeptide” also includes an oligopeptide. Further, any referenceto a “peptide” includes polypeptides, oligopeptides, and proteins. Eachdifferent arrangement of amino acids forms different polypeptides orproteins. The number of polypeptides—and hence the number of differentproteins—that can be formed is practically unlimited.

Since the peptide bond is the sole linkage between amino acids, allpeptides, polypeptides or proteins have defined termini conventionallyreferred to as the “N-terminus” or “N-terminal” residue and the“C-terminus” or “C-terminal residue”. The N-terminal residue bears afree amino group, whereas the C-terminal residue bears a free carboxylgroup.

All sequences of consecutive amino acids accordingly have an orientationN-terminal to C-terminal. Where fusion proteins are constituted ordiffering domains are connected within a protein species their relativeorientation may be described as “N-terminal” or “C-terminal”.

The term “fusion protein” is used herein to refer to a protein moleculecomprising two or more functionally distinct protein domains within asingle polypeptide chain. The protein moieties in the fusion protein maybe directly coupled or may be joined via a linker peptide.

A “linker” or “linker peptide” refers herein to a peptide segmentjoining two moieties of fusion protein. An example of a linker peptideis provided by the amino acid sequence (G)₄S(G)₄S(G)₃SG (SEQ ID NO: 5).However, also other linker peptides, preferably having 4-20 amino acidresidues can be used according to the invention. The fusion proteins ofthe present invention contain such a linker but not all fusion proteinscontain a linker.

Fusion proteins are commonly produced by means of recombinant DNAtechniques and as such can be considered artificial proteins having nodirect counterparts in nature (natural fusion proteins can arise, forexample via chromosomal translocation, but are not considered here). Anexample of a fusion protein is a fusion in which an immunoglobulin Fcregion is placed at the N-terminus of another protein such as TPO. Sucha fusion is termed an “Fc-X” fusion, where X is a ligand (such as TPO)and Fc-X proteins have a number of distinctive, advantageous biologicalproperties. In particular, whereas such fusion proteins can still bindthe relevant Fc receptors on cell surfaces, when the ligand binds to itsreceptor, the orientation of the Fc region is altered such thatantibody-dependent cell-mediated cytotoxicity and complement fixationare activated by the sequences present in the Fc domain.

The term “immunoglobulin” is used herein to refer to a proteinconsisting of one or more polypeptides substantially encoded byimmunoglobulin genes. The recognised immunoglobulin genes include thekappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, IgG4), sigma, epsilon,and μ constant region genes and in nature multiple immunoglobulinvariable region genes.

The term Fc is used herein to refer to an immunoglobulin heavy chainconstant region domain.

The term “T-cell epitope” means according to the understanding of thisinvention an amino acid sequence which is able to bind MEC class II,able to stimulate T-cells and/or also to bind (without necessarilymeasurably activating) T-cells in complex with MHC class II.

Reference to “substantially non-immunogenic” or “reduced immunogenicpotential” includes reduced immunogenicity compared to a parent proteinor to a fusion protein containing the wild-type (WT) or native aminoacid sequences of the test moiety.

The term “immunogenicity” includes an ability to provoke, induce orotherwise facilitate a humoral and or T-cell mediated response in a hostanimal and in particular where the “host animal” is a human.

The terms “T-cell assay” and “immunogenicity assay” concern ex vivomeasures of immune reactivity. As such these involve a test immunogene.g. a protein or peptide being brought into contact with live humanimmune cells and their reactivity measured. A typical parameter ofinduced reactivity is proliferation. The presence of suitable controldeterminations are critical and implicit in the assay.

“Time course assay” refers to a biological assay such as a proliferationassay in which determinations of activity are made sequentially over aperiod of time. In the present context, a “time course T-cell assay”,refers to the determination of T-cell proliferation in response to atest immunogen (peptide) at multiple times following exposure to thetest immunogen. The terms “time course T-cell assay” and “time courseimmunogenicity assay” may be used interchangeably herein.

One conventional way in which T-cell assays are expressed is by use of a“stimulation index” or “SI”. The stimulation index (SI) isconventionally derived by division of the proliferation score (e.g.counts per minute of radioactivity if using for example ³H-thymidineincorporation) measured to a test immunogen such as a peptide by thescore measured in cells not contacted with a test immunogen. Testimmunogens (peptides) which evoke no response give SI=1.0 although inpractice SI values in the range 0.8-1.2 are unremarkable. The inventorshave established that in the operation of such immunogenicity assays, astimulation index equal to or greater than 2.0 is a useful measure ofsignificant induced proliferation.

PBMC means peripheral blood mononuclear cells in particular as obtainedfrom a sample of blood from a donor. PBMC are readily isolated fromwhole blood samples using a density gradient centrifugation techniquewell understood in the art and comprise predominantly lymphocytes (B andT cells) and monocytes. Other cell types are also represented.

“Relative activity” means according the present context activitymeasured for a test protein in any single assay expressed relative tothe activity measured for a positive control protein in an identicalassay and usually conducted in parallel. Thus if the test protein andthe control protein have the same measured activity the relativeactivity is said to be 1.

A “proliferation assay” according to the present context means abiological assay able to provide a reading of the functional capabilityof the test protein. In the present instance this means the ability of agiven TPO mutein or TPO fusion protein to evoke a specific measurableproliferative response in a live cell. Particularly suitableproliferation assays are exemplified herein using TF-1 cells or M0-e7cells. Other cells and assay formats can be contemplated to also providequantitative estimations of specific activity of the test molecules andpermit ED₅₀ determinations.

In another aspect, the present invention relates to nucleic acidsencoding modified TPO entities. Such nucleic acids are preferablycomprised within an expression vector. The control sequences that aresuitable for prokaryotes, for example, include a promoter, optionally anoperator sequence, and a ribosome binding site. Eukaryotic cells areknown to utilise promoters, enhancers and polyadenylation signals. Suchnucleic acids in general comprise a selection means typically anadditional gene encoding a protein able to provide for the survival ofthe host cell. An example of such a selection gene is the beta-lactamasegene suitable for some E. coli host cells and this and others are wellknown in the art [“Molecular Cloning: A Laboratory Manual”, secondedition (Sambrook et al., 1989); “Gene Transfer Vectors for MammalianCells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols inMolecular Biology” (F. M. Ausubel et al., eds., 1987)].

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apre-sequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a pre-protein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in the same reading frame.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

In some embodiments the expression vector comprises a nucleic acidsequence encoding a TPO variant operably linked to an expression controlsequence. In various embodiments the expression vector comprises anucleic acid sequence encoding a TPO protein selected from the groupcomprising inclusively M1 to M68. Such an expression vector willcomprise at least the TPO encoding domain of one of the said proteinsoperably linked with suitable expression control and selectionsequences. Such an expression vector would include degenerate versionsof the nucleic acid wherein degeneracy in relation to polynucleotidesrefers to the fact well recognised that in the genetic code many aminoacids are specified by more than one codon. The degeneracy of the codeaccounts for 20 different amino acids encoded by 64 possible tripletsequences of the four different bases comprising DNA.

Another aspect of the present invention is a cultured cell comprising atleast one of the above-mentioned vectors.

A further aspect of the present invention is a method for preparing themodified TPO comprising culturing the above mentioned cell underconditions permitting expression of the TPO from the expression vectorand purifying the TPO from the cell.

In a yet further aspect, the present invention relates to methods fortherapeutic treatment of humans using the TPO compositions. Foradministration to an individual, any of the modified compositions wouldbe produced to be preferably at least 80% pure and free of pyrogens andother contaminants. It is further understood that the therapeuticcompositions of the TPO proteins may be used in conjunction with apharmaceutically acceptable excipient. The pharmaceutical compositionsaccording to the present invention are prepared conventionally,comprising substances that are customarily used in pharmaceuticals, e.g.Remington's Pharmaceutical Sciences, (Alfonso R. Gennaro ed. 18^(th)edition 1990), including excipients, carriers adjuvants and buffers. Thecompositions can be administered, e.g. parenterally, enterally,intramuscularly, subcutaneously, intravenously or other routes useful toachieve an effect. Conventional excipients include pharmaceuticallyacceptable organic or inorganic carrier substances suitable forparenteral, enteral and other routes of administration that do notdeleteriously react with the agents. For parenteral application,particularly suitable are injectable sterile solutions, preferably oilor aqueous solutions, as well as suspensions, emulsions or implants,including suppositories. Ampules are convenient unit dosages. Thepharmaceutical preparations can be sterilised and, if desired, mixedwith stabilisers, wetting agents, emulsifiers, salts for influencingosmotic pressure, buffers or other substances that do not reactdeleteriously with the active compounds.

The major embodiments of the present invention are encompassed by theTPO protein sequences M1-M67 and the fusion protein sequences F-M1 toF-M67, or F-L-M1 to F-L-M67, or F1-L1-M1 to F1-L1-M67. The proteins arefusion proteins of the type “Fc-X” wherein X in this present instancecomprise TPO muteins. The TPO proteins are expressed in mammaliancell-lines as a C-terminal fusion partner, linked to the Fc unit ofhuman IgG₄. The TPO sequence is fused preferably to the C-terminus of ahinge modified/C_(H)2/C_(H)3 Fc region of human IgG₄ via a 15 amino acidflexible linker between the C-terminus of the C_(H)3 and the N-terminusof TPO. The TPO domain comprises only residues 1-174 of the nativecounterpart. The amino acid sequence of the linker was as follows:(G)₄S(G)₄S(G)₃SG (SEQ ID NO: 5). The expressed fusion protein had astoichiometry of (hinge-C_(H)2—C_(H)3-linker-TPO₍₁₋₁₇₄₎)₂.

Human Fc-gamma 4 was used as the fusion partner in all preferredmolecules, but it can be readily recognised that in principle otherisotypes could equally be used. In the present instance, immune effectorfunctions are not desirable for a therapeutic TPO molecule. In contrastto some other human Fc isotypes, the Fc-gamma 4 isotype does not supportcomplement activation and antibody-dependent cell-mediated cytotoxicity(ADCC) and was therefore selected for as the most preferred fusionpartner.

Where the “Fc-X” approach has been used in other molecules, such as forexample Fc-IL10 and IL10-Fc, the in vivo half-life in mice was extendedfrom minutes to greater than 30 hours [Lo K-M, et al (1998) ProteinEngineering; 11: 495-500; Gillies S D, et al (1999) Cancer Research; 59:2159-2166; Zheng X. X. et al (1995) Journal of Immunology; 154:5590-5600]. Similarly, where an “X-Fc” molecule has been used as atherapeutic in humans, the serum half-life is recorded at 3 days(Korth-Bradley J M, et al (2000) Annals of Pharmacotherapy; 34:161-164].

The inventors have provided TPO fusion proteins that show increasedactivity compared to the fusion proteins containing the wild-type (WT)TPO moiety. The “WT” or “native” fusion proteins constructed herein hasbeen designated clone ID 00 (M68, F-M68. F-L-M68, F1-L1-M68).

Using a proliferation assay in TF-1 cells, the native fusion protein hasbeen found to have an ED₅₀ value of around 12.0 ng/ml in somedeterminations and when using M0-7e cells, around 25.0 ng/ml in somedeterminations

By contrast, it has been somewhat surprisingly found that a mostpreferred molecule of the invention (M1, F-M1. F-L-M1, F1-L1-M1) has anED₅₀ value in TF-1 cells of around 11.5 ng/ml in some determinations andwhen using M0-7e cells an ED₅₀ of around 18.0 ng/ml. Given that thismolecule is a TPO mutein, these results indicate that the changes to thesequence have had a beneficial effect on direct functional activity.

Enhanced potency in the molecule attributed to the dimeric nature of theprotein by virtue of the Fc-domain is demonstrated by comparison of theED₅₀ values found using the full size (non-Fc linked) human TPO moleculein TF-1 and E0-7e based proliferation assays. In the present studies,monomeric full-length recombinant human (r-hTPO) TPO achieves an ED₅₀ ofaround 29.5 ng/ml using TF-1 cells and around 70.0 ng/ml using M0-7ecells. A most preferred molecule of the invention therefore demonstratesapproximately between 2.5-4.0 fold enhanced activity over r-hTPO.

A further example of an especially preferred molecule of the inventionwith significantly enhanced activity provided by the TPO muteincontaining the substitiution set M55K, A60R, V161A (M67, F-M67. F-L-M67,F1-L1-M67). This protein is highly potent in the TF-1 assay with arelative activity of 0.4. This molecule is therefore more active eventhan a TPO fusion protein with a WT TPO domain (M68, F-M68.F-L-M68,F1-L1-M68).

Although the M55K, A60R, V161A (M67,F-M67.F-L-M67,F1-L1-M67) mutein isclearly a highly potent molecule, this mutein is not as active as themutein comprising only the M55K and A60R substitutions (M66,F-M66.F-L-M66,F1-L1-M68). This mutein demonstrates a relative activityof 0.2 in the TF-1 assay.

Accordingly therefore, the TPO proteins M1, M66 and M67 including theirdifferent fusion structures as indicated above and below, are especiallypreferred molecules of the invention.

The TPO muteins of the present were constructed to be less immunogenicthan the parental molecule. The design of individual muteins wasdirected from immunological considerations as well as functionalactivity data. Two regions of immunological importance within theN-domain of the molecule were defined using screening assays involvinguse of PBMC preparations from healthy donor subjects. This approach hasproven to be a particularly effective method for the identification suchbiologically relevant immunogenic peptides and is disclosed herein as anembodiment of the invention. In the present study, the method hasinvolved the testing of overlapping TPO-derived peptide sequences in ascheme so as to scan and test the TPO sequence comprising residues1-177. Such a scan required synthesis and use of 55 peptides each of 15residues in length. The synthetic peptides were tested for their abilityto evoke a proliferative response in human T-cells cultured in vitro.Where this type of approach is conducted using naïve human T-cells takenfrom healthy donors, the inventors have established that a stimulationindex equal to or greater than 2.0 is a useful measure of inducedproliferation.

Two epitope regions were identified in these studies. Region 1encompasses TPO residues 49-75 and comprises the sequence:GEWKTQMEETKAQDILGAVTLLLEGVM (SEQ ID NO: 2). Region 2 encompasses TPOresidues 157-171 and comprises the sequence: PTTAVPSRTSLVLTL (SEQ ID NO:3).

The R1 and R2 peptide sequences represent the critical informationrequired for the construction of modified TPO molecules in which one ormore of these epitopes is compromised. Equally, The R1 and R2 peptidesequences represent the critical information required for the productionof tolerogenic peptides. Epitope regions R1 and R2 are each embodimentsof the invention.

Under the scheme of the present, the epitopes are compromised bymutation to result in sequences no longer able to function as T-cellepitopes. It is possible to use recombinant DNA methods to achievedirected mutagenesis of the target sequences and many such techniquesare available and well known in the art. Broadly, the TPO muteins hereinwere constructed containing mutations within the two identifiedimmunogenic regions R1 and R2. Individual residues were targeted basedupon the known binding properties of HLA-DR molecules in that they havean almost exclusive preference for a hydrophobic amino acid in pocket 1and that this is the most important determinant of peptide binding[Jardetzky, T. S. et al (1990), EMBO J. 9: 1797-1803; Hill, C. M. et al(1994) J. Immunol. 152: 2890-2898]. Exhaustive mutational analysisidentified those residues within these regions that could be alteredwithout adversely affecting the activity of the fusion protein. Choiceof alternate residue was guided comparison to other TPO proteins fromother species. Buried residues were replaced with either alanine orsimilar sized non-hydrophobic residues whereas exposed residues werescanned with all possible non-hydrophobic alternatives.

The general method of the present invention leading to the modified TPOcomprises the following steps:

(a) determining the amino acid sequence of the polypeptide or partthereof;

(b) identifying one or more potential T-cell epitopes within the aminoacid sequence of the protein by any method including determination ofthe binding of the peptides to MHC molecules using in vitro or in silicotechniques or biological assays;

(c) designing new sequence variants with one or more amino acids withinthe identified potential T-cell epitopes modified in such a way tosubstantially reduce or eliminate the activity of the T-cell epitope asdetermined by the binding of the peptides to MHC molecules using invitro or in silico techniques or biological assays. Such sequencevariants are created in such a way to avoid creation of new potentialT-cell epitopes by the sequence variations unless such new potentialT-cell epitopes are, in turn, modified in such a way to substantiallyreduce or eliminate the activity of the T-cell epitope; and(d) constructing such sequence variants by recombinant DNA techniquesand testing said variants in order to identify one or more variants withdesirable properties according to well known recombinant techniques.

Taken together, the inventors have been able to define improved TPOproteins which can be depicted by the following structure (M):

SPAPPACDLRVLSKLLRDSHVLHSRLSQCPEVHPLPTPVLLPAVDFSLGX¹X²KTQX³EEX⁴KX⁵X⁶DX⁷LGAX⁸TX⁹LX¹⁰X¹¹GVMAARGQLGPTCLSSLLGQLSGQVRLLLGALQSLLGTQLPPQGRTTAHKDPNAIFLSFQHLLRGKVRFLMLVGGSTLCVRRAPPTTAX¹²X¹³SRTSLVLTLNEL (SEQ ID NO: 1),wherein

-   -   X¹ is A, E;    -   X² is S, W;    -   X³ is A or T or K, S or M;    -   X⁴ is A, T;    -   X⁵ is R, A;    -   X⁶ is A or T or Q;    -   X⁷ is A or T or I;    -   X⁸ is A or T or V;    -   X⁹ is A or T or S or L;    -   X¹⁰ is A or L;    -   X¹¹ is A or S or E;    -   X¹² is N or A or T or R or E or D or G or H or P or K or Q or V;    -   X¹³ is A or P,    -   and whereby simultaneously X¹=E, X²=W, X³=M, X⁴=T, X⁵=A, X⁶=Q,        X⁷=I, X⁸=V, X⁹=L, X¹⁰=L, X¹¹=E, X¹²=V and X¹³=P are excluded,        or, alternatively, fusion proteins of the structure:        F-(L)n-M,        wherein M has the meaning as specified above, F is an        immunoglobulin heavy chain constant region, preferably an Fc        portion, and L is an optional linker molecule (n=0, 1),        preferably a peptide linker having 4-20 amino acid residues.        Preferably the Fc region derives from human IgG4 an may be        linked at its N-terminal to a hinge region, which may be        modified in order to reduce immunogenicity or to improve other        desired properties.

The following, figures, sequence listing and examples are provided toaid the understanding of the present invention. It is understood thatmodifications can be made in the procedures set fourth without departingfrom the spirit of the invention.

DESCRIPTION OF THE SEQUENCES

To aid the understanding of the invention, Table 1 below sets out adescription of the fusion protein TPO muteins. The derivation andproperties of these proteins are also more fully disclosed in theexamples. In Table 1 the column heading labeled “Substitution(s)” refersto substitutions in SEQ ID NO: 4, i.e., native human TPO.

TABLE 1 F1-L1-M Clone ID Substitution(s)* Sequence No. 37101 A60R, V161AM1 1394867 I63A, V67T, V161N, P162A M2 12394867 I63T, V67A, V161N, P162AM3 1374972 M55A, I63A, V67A, V161A M4 1374973 M55T, I63A, V67A, V161A M512484973 M55T, I63T, V67A, V161A M6 1374968 Q61A, I63A, V67A, V161A M712484968 Q61A, I63T, V67A, V161T M8 124849 I63T, V67A, V161T M9 123749I63T, V67A, V161A M10 13749 I63A, V67A, V161A M11 14849 I63A, V67A,V161T M12 124860 I63T, V67T, V161T M13 3849 V67A, V161R M14 4849 V67A,V161T M15 3760 V67T, V161A M16 4860 V67T, V161T M17 3749 V67A, V161A M184249 V67A, V161E M19 1248 I63T, V161T M20 1238 I63T, V161R M21 1242I63T, V161E M22 1237 I63T, V161A M23 149 I63A, V67A M24 160 I63A, V67TM25 1249 I63T, V67A M26 137 I63A, V161A M27 142 I63A, V161E M28 138I63A, V161R M29 148 I63A, V161T M30 6063 E50A, V67T M31 163 E50A, I63AM32 1263 E50A, I63T M33 4263 E50A, V161E M34 37 V161A M35 40 V161D M3642 V161E M37 43 V161G M38 44 V161H M39 39 V161N M40 46 V161P M41 45V161K M42 41 V161Q M43 38 V161R M44 48 V161T M45 49 V67A M46 60 V67T M471 I63A M48 12 I63T M49 68 Q61A M50 69 Q61T M51 72 M55A M52 102 M55K M5374 M55S M54 73 M55T M55 100 T58A M56 35 W51S M57 63 E50A M58 77 L69A M5978 L69S M60 79 L69T M61 83 L71A M62 86 E72A M63 87 E72S M64 101 A60R M65101102 M55k, A60R M66 37101102 M55k, A60R, V161A M67 00 WT M68 *Theresidue numbering for the TPO substitutions commences from residue 1 ofthe TPO reading frame and is independent of any Fc component.

M1-M67 (modified human TPO, truncated form 1-174) M1 (SEQ ID NO: 6):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K R Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M2(SEQ ID NO: 7):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D A L G A T T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A N A S R T S L V L T L N E L M3(SEQ ID NO: 8):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D T L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A N A S R T S L V L T L N E L M4(SEQ ID NO: 9):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q A E E T K A Q D A L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M5(SEQ ID NO: 10):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q T E E T K A Q D A L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M6(SEQ ID NO: 11):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q T E E T K A Q D T L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A T S R T S L V L T L N E L M7(SEQ ID NO: 12):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A A D A L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M8(SEQ ID NO: 13):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A A D T L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A T P S R T S L V L T L N E L M9(SEQ ID NO: 14):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D T L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A T P S R T S L V L T L N E L M10(SEQ ID NO: 15):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D T L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M11(SEQ ID NO: 16):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D A L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M12(SEQ ID NO: 17):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D A L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M13(SEQ ID NO: 18):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D T L G A T T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M14(SEQ ID NO: 19):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M15(SEQ ID NO: 20):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M16(SEQ ID NO: 21):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A T T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M17(SEQ ID NO: 22):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A T T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M18(SEQ ID NO: 23):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M19(SEQ ID NO: 24):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A E P S R T S L V L T L N E L M20(SEQ ID NO: 25):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D T L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A T P S R T S L V L T L N E L M21(SEQ ID NO: 26):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D T L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A R P S R T S L V L T L N E L M22(SEQ ID NO: 27):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D T L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A E P S R T S L V L T L N E L M23(SEQ ID NO: 28):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D T L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M24(SEQ ID NO: 29):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D A L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M25(SEQ ID NO: 30):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D A L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M26(SEQ ID NO: 31):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D T L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M27(SEQ ID NO: 32):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D A L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M28(SEQ ID NO: 33):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D A L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A E P S R T S L V L T L N E L M29(SEQ ID NO: 34):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D A L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A R P S R T S L V L T L N E L M30(SEQ ID NO: 35):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D A L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A T P S R T S L V L T L N E L M31(SEQ ID NO: 36):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A T T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M32(SEQ ID NO: 37):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D A L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M33(SEQ ID NO: 38):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D T L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M34(SEQ ID NO: 39):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G A W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A E P S R T S L V L T L N E L M35(SEQ ID NO: 40):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M36(SEQ ID NO: 41):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A D P S R T S L V L T L N E L M37(SEQ ID NO: 42):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A E P S R T S L V L T L N E L M38(SEQ ID NO: 43):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A G P S R T S L V L T L N E L M39(SEQ ID NO: 44):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A H P S R T S L V L T L N E L M40(SEQ ID NO: 45):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A N P S R T S L V L T L N E L M41(SEQ ID NO: 46):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A P P S R T S L V L T L N E L M42(SEQ ID NO: 47):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A K P S R T S L V L T L N E L M43(SEQ ID NO: 48):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A Q P S R T S L V L T L N E L M44(SEQ ID NO: 49):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A R P S R T S L V L T L N E L M45(SEQ ID NO: 50):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A T P S R T S L V L T L N E L M46(SEQ ID NO: 51):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A A T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M47(SEQ ID NO: 52):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A T T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M48(SEQ ID NO: 53):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D A L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M49(SEQ ID NO: 54):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D T L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M50(SEQ ID NO: 55):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A A D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M51(SEQ ID NO: 56):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A T D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M52(SEQ ID NO: 57):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q A E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M53(SEQ ID NO: 58):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q K E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M54(SEQ ID NO: 59):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q S E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M55(SEQ ID NO: 60):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q T E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M56(SEQ ID NO: 61):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E A K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M57(SEQ ID NO: 62):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E S K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M58(SEQ ID NO: 63):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G A W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M59(SEQ ID NO: 64):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T A LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M60(SEQ ID NO: 65):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T S LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M61(SEQ ID NO: 66):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T T LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M62(SEQ ID NO: 67):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M63(SEQ ID NO: 68):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M64(SEQ ID NO: 69):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M65(SEQ ID NO: 70):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K R Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M66(SEQ ID NO: 71):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q K E E T K R Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L M67(SEQ ID NO: 72):S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q K E E T K R Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A A P S R T S L V L T L N E L M68(wild-type human TPO, truncated form 1-174)S P A P P A C D L R V L S K L L R D S H V L H S R L S Q C P E V H P LP T P V L L P A V D F S L G E W K T Q M E E T K A Q D I L G A V T L LL E G V M A A R G Q L G P T C L S S L L G Q L S G Q V R L L L G A L QS L L G T Q L P P Q G R T T A H K D P N A I F L S F Q H L L R G K V RF L M L V G G S T L C V R R A P P T T A V P S R T S L V L T L N E L F1(Fc domain of human IgG4 including modified hinge region)E P K S S D K T H T C P P C P A P E F L G G P S V F L F P P K P K D T L M IS R T P E V T C V V V D V S Q E D P E V Q F N W Y V D G V E V H N A K T K PR E E Q F N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K G L PS S I E K T I S K A K G Q P R E P Q V Y T L P P S Q E E M T K N Q V S L T CL V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F FL Y S K L T V D K S R W Q Q G N I F S C S V M H E A L H N H Y T Q K S L S LS P G A L1 (Linker peptide) G G G G S G G G G S G G G S G

Examples of fusion proteins F-M of the present invention (in which F isany immunoglobulin heavy chain constant region and M is a modified TPOsequence of the invention) include the following: F - M1, F - M2, F -M3, F - M4, F - M5, F - M6, F - M7, F - M8, F - M9, F - M10, F - M11,F - M12, F - M13, F - M14, F - M15, F - M16, F - M17, F - M18, F - M19,F - M20, F - M21, F - M22, F - M23, F - M24, F - M25, F - M26, F - M27,F - M28, F - M29, F - M30, F - M31, F - M32, F - M33, F - M34, F - M35,F - M36, F - M37, F - M38, F - M39, F - M40, F - M41, F - M42, F - M43,F - M44, F - M45, F - M46, F - M47, F - M48, F - M49, F - M50, F - M51,F - M52, F - M53, F - M54, F - M55, F - M56, F - M57, F - M58, F - M59,F - M60, F - M61, F - M62, F - M63, F - M64, F - M65, F - M66, F - M67.

Examples of fusion proteins F - L - M of the present invention (in whichF is any immunoglobulin heavy chain constant region, L is a linkerpeptide, and M is a modified TPO sequence of the invention) include thefollowing: F - L - M1, F - L - M2, F - L - M3, F - L - M4, F - L - M5,F - L - M6, F - L - M7, F - L - M8, F - L - M9, F - L - M10, F - L -M11, F - L - M12, F - L - M13, F - L - M14, F - L - M15, F - L - M16,F - L - M17, F - L - M18, F - L - M19, F - L - M20, F - L - M21, F - L -M22, F - L - M23, F - L - M24, F - L - M25, F - L - M26, F - L - M27,F - L - M28, F - L - M29, F - L - M29, F - L - M30, F - L - M31, F - L -M32, F - L - M33, F - L - M34, F - L - M35, F - L - M36, F - L - M37,F - L - M38, F - L - M39, F - L - M40, F - L - M41, F - L - M42, F - L -M43, F - L - M44, F - L - M45, F - L - M46, F - L - M47, F - L - M48,F - L - M49, F - L - M50, F - L - M51, F - L - M52, F - L - M53, F - L -M54, F - L - M55, F - L - M56, F - L - M57, F - L - M58, F - L - M59,F - L - M60, F - L - M61, F - L - M62, F - L - M63, F - L - M64, F - L -M65, F - L - M66, F - L - M67.

Examples of fusion proteins F1 - L1 - M of the present invention (inwhich F1 is the Fc portion from human IgG4, L1 is the peptide linkerdescribed above, and M is a modified TPO sequence of the invention)include the following: F1 - L1 - M1, F1 - L1 - M2, F1 - L1 - M3, F1 -L1 - M4, F1 - L1 - M5, F1 - L1 - M6, F1 - L1 - M7, F - L - M8, F1 - L1 -M9, F1 - L1 - M10, F1 - L1 - M11, F1 - L1 - M12, F1 - L1 - M13, F1 -L1 - M14, F1 - L1 - M15, F1 - L1 - M16, F1 - L1 - M17, F1 - L1 - M18,F1 - L1 - M19, F1 - L1 - M20, F1 - L1 - M21, F1 - L1 - M22, F1 - L1 -M23, F1 - L1 - M24, F1 - L1 - M25, F1 - L1 - M26, F1 - L1 - M27, F1 -L1 - M28, F1 - L1 - M29, F1 - L1 - M29, F1 - L1 - M30, F1 - L1 - M31,F1 - L1 - M32, F1 - L1 - M33, F1 - L1 - M34, F1 - L1 - M35, F1 - L1 -M36, F1 - L1 - M37, F1 - L1 - M38, F1 - L1 - M39, F1 - L1 - M40, F1 -L1 - M41, F1 - L1 - M42, F1 - L1 - M43, F1 - L1 - M44, F1 - L1 - M45,F1 - L1 - M46, F1 - L1 - M47, F1 - L1 - M48, F1 - L1 - M49, F1 - L1 -M50, F1 - L1 - M51, F1 - L1 - M52, F1 - L1 - M53, F1 - L1 - M54, F1 -L1 - M55, F1 - L1 - M56, F1 - L1 - M57, F1 - L1 - M58, F1 - L1 - M59,F1 - L1 - M60, F1 - L1 - M61, F1 - L1 - M62, F1 - L1 - M63, F1 - L1 -M64, F1 - L1 - M65, F1 - L1 - M66, F1 - L1 - M67.

A fusion protein with wild-type human TPO (M68) is: F1 - L1 -M68.

DESCRIPTION OF THE FIGURES

FIG. 1:

Identification of T cell epitopes in TPO (1-174). (A) 20 healthy donorswere tested for reactivity with 55 overlapping (by 12 amino acids) 15mer peptides derived from the TPO sequence. Donors that responded topeptides with an SI>2 were analyzed further by plotting the frequency ofdonor responses to each peptide. Prominent regions of immunogencity arelabeled according to the amino acid residue number in the TPO linearsequence and were determined by peptides that induced responses in 10%of donors; however, borderline responses where individual SI values>1.95(two tone black and white bars) were achieved and if two (or more)adjacent peptides induced responses in 5% of donors (Region 1). (B) Themature sequence of TPO (SEQ ID NO: 4) with regions of immunogenicityboxed and highlighted in bold.

FIG. 2:

Frequency of observed responses with an SI>2 at any time point fromcohorts of 20 healthy donors to either wild type region 1 and modifiedregion 1 peptides (A); or wild type region 2 and modified region 2peptides (B).

FIG. 3:

Immunogenicity of TPO variant peptides. Two cohorts of 20 healthy donorswere used to test the immunogenicity of either wild type region 1 andmodified region 1 peptides (A) or wild type region 2 and modified region2 peptides (B). Proliferation of PBMC was assessed by tritiatedthymidine incorporation on days 6, 7, 8 and 9 post-stimulation andstimulation indexes were calculated.

FIG. 4:

Mutein clone ID 102 (M53/F1-L1-M53) comprising single amino acidsubstitution M55K shows greater activity in a functional assay thaneither the WT counterpart or a control TPO preparation lacking the Fcdomain. Functional activity is plotted as CPM measued using theproliferation assay (Example 4) versus concentration of TPO proteinadded. Proliferation was measured in TF-1 cells using culturesupernatants from TPO mutein and TPO WT transfected HEK.293 cells.Supernatants were quantified by Fc ELISA and diluted to 320 ng/ml. Theactivity was titrated in 2 fold serial dilutions.

EXPERIMENTAL EXAMPLES Example 1

Construction of Fc-TPO Muteins

The modified TPO proteins of the present invention were made usingconventional recombinant DNA techniques. The N-terminal domain of theprotein was cloned comprising residues 1-174. The coding sequence forTPO (1-174) was cloned from human human liver cDNA library using PCR.The wild-type gene was used both as a control reagent and a templatefrom which to derive modified TPO proteins by site directed mutagenesis.WT and modified genes were inserted into a modified version of theexpression vector pdC-huFc [K.-M. Lo et al., (1998) Protein Eng11:495-500]. The TPO gene was excised with XmaI and XhoI and cloned intoa similarly cut preparation of the vector which had been modified suchthat the TPO sequence is fused to the C-terminus of a hingemodified/C_(H)2/C_(H)3 Fc region of human IgG₄ via a 15 amino acidflexible linker between the C-terminus of the C_(H)3 and the N-terminusof TPO₍₁₋₁₇₄₎. The amino acid sequence of the linker was as follows:(G)₄S(G)₄S(G)₃SG (SEQ ID NO: 5). The expressed fusion protein had astoichiometry of (hinge-C_(H)2—C_(H)3-linker-TPO₍₁₋₁₇₄₎)₂. The finalconstruct used in this study was designated Fc-gamma 4-linker-TPO (cloneID 00, M68/F1-L1-M68).

DNA sequencing was conducted on all constructs. This was diligentlyperformed to confirm introduction of desired substitutions and establishthat no extraneous (undesired) substitutions had been introduced forexample by PCR error.

Variants of TPO₍₁₋₁₇₄₎ linked to the Fc portion of human IgG₄ wereconstructed containing mutations within the two immunogenic regions ofthis domain of the protein. Desired substitutions were introduced intothe TPO sequence by overlap PCR using HiFi Expand polymerase. Cycles ofmutational analysis involving construction and function testingidentified those residues within these regions that could be alteredwithout adversely affecting the activity of the Fc-linked protein. Theproliferation assay as described herein (see Example 4) was the mainscreening tool in this aspect, a total of 667 different muteins in TPOwere identified with positive functional activity (Table 2).

Example 2

Transfection and Purification of Fusion Proteins

Transient transfections were done using HEK293 (ATCC# CRL-1573) cellsand Lipofectamine 2000 (Invitrogen, Paisley, UK) as described by themanufacturer. Stable transfectants were also made in HEK293 cells andselected in media containing increasing concentrations of methotrexate.All cell-lines were maintained in DMEM plus 10% FBS with antibiotics andantimycotics. Fusion proteins were purified via Prosep-A chromatographyfollowed by size exclusion chromatography (SEC). Briefly, 1 ml Prosep®-Acolumns (Millipore, Watford, UK) were equilibrated in PBS pH 7.4 beforebeing loaded with 0.2 μM filtered cell-culture supernatants (up to 500ml) that had been pH adjusted with 1/20 vols. 1M Tris-HCl pH 7.4. Thecolumn was washed with 50 ml PBS pH 7.4 and the fusion protein elutedwith 0.1M citrate buffer pH 3.0 and 0.9 ml fractions collected. Thefractions were immediately neutralized with 0.1 ml 1M Tris-HCl pH 8.0.SEC was done with Superdex 200 (Amersham Pharmacia, Amersham, UK) in a3.2/30 column equilibrated and run in PBS pH 7.4 containing 0.1% Tween80. Fractions spanning the major peak were pooled and fusion proteinsquantified using molar extinction coefficients at 280 nm calculatedusing Lasergene™ software (Dnastar, Madison, Wis., USA). Theconcentrations were confirmed using a BCA protein assay (Pierce,Chester, UK).

Example 3

Quantitation of Fusion Proteins in Cell-Culture Supernatants

Fusion proteins were quantified by detecting the amount of human IgG₄ Fcin an ELISA format as follows: ELISA plates (Dynex Immulon4) were coatedwith a mouse monoclonal anti-human IgG Fc specific antibody at adilution of 1/1500 in PBS pH7.4, 100 μl/well, for 2 h at 37° C. Theplate was washed ×4 with 100 μl/well PBS/0.05% Tween 20. Human IgGstandards (The Binding Site, Birmingham, UK) were diluted to 2 μg/ml inPBS/2% BSA and duplicate two-fold dilutions made vertically down theplate. Test samples were diluted 1/100 and 1/500 in PBS/2% BSA andassayed in duplicate. The plate was incubated for 1 h at roomtemperature and washed as before. Detection was done using 100 μl/wellgoat anti-human IgG Fc-specific peroxidase conjugate (The Binding Site,Birmingham, UK) at a dilution of 1/1000 in PBS, the plate washed asbefore and colour developed using SigmaFast OPD, 100 μl/well (Sigma,Poole, UK). The colour reaction was stopped by the addition of 50 μl 2Msulphuric acid and the absorbance measured at 492 nm in an Anthos HTIIplate reader.

Example 4

Functional Activity of Fc-TPO Muteins

The functional activity of the Fc-TPO proteins was compared usingproliferation assay using either the erythro leukaemia cell line TF-1,or the megakaryocytic cell line M0-7e [Avanzi G C et al (1988) BritishJournal of Haematology; 69: 359-366; Jagerschmidt A, et al (1998)Biochemical Journal; 333: 729-734 and Quentmeier H, et al (1996)Leukemia; 10:297-310]. Cells were grown in RPMI-1640 (Invitrogen,Paisley, UK) with 10% serum supreme (Biowhittaker, Wokingham, UK),penicllin/streptomycin (Invitrogen, Paisley, UK), and GM-CSF (Peprotech,London, UK) at 2 ng/ml for TF-1 and 10 ng/ml for M0-7e.

For the assay, exponentially growing TF-1 or M0-7e cells were seededinto 96 well plates at a concentration of 2×10⁴ cells per well, in assaymedium supplemented with increasing amounts of conditioned medium fromHEK293 cells transfected with the vector containing the protein ofinterest. Recombinant TPO (Peprotech) with a specific activity of 1×10⁶units/mg was used as an additional positive control reagent in theseassays. Assays were performed at TPO concentrations ranging from 0 to320 ng/ml, with duplicate serial doubling dilutions of standard r-hTPO(Peprotech) and triplicate serial doubling dilutions of test proteinvariants in antibiotic free media, were made horizontally across a “U”bottomed 96 well plate. The plates were incubated for 96 h at 37° C. andthen 1 μCi of [³H]-thymidine added over night. Cells were harvested ontofilter maps and then solid scintillant melted onto the mat using a hotplate. Counts per minute (CPM) were then measured using a MicroBeta TriLux scintillation counter. CPM vs TPO concentration was plotted and anEC₅₀ value determined.

A total of 67 different TPO variants demonstrated positive activity inthe proliferation assay. Positive activity was taken to be a relativeactivity value of less than 10. Relative activity was determined bydividing the ED₅₀ value derived for the protein of interest by the ED₅₀value derived for the control (WT) TPO fusion protein (M66/F1-L1-M66).Of these active proteins, 31 were muteins comprising a single amino acidsubstitution; 23 comprised 2 amino acid substitutions, 7 comprised 3amino acid substitutions and 7 comprised four amino acid substitutions.The sequence of each of these active TPO muteins is provided in M1 - M67(F1-L1-M67). The relative activities of each functioning mutein areprovided in Table 2. In Table 2, the heading labled “Substitution(s)”refers to an amino acid residue substitution in SEQ ID NO: 4 (humanTPO).

TABLE 2 Activity of FC-TPO variants Relative Activity* F1-L1-M(proliferation in Clone ID Substitution(s) sequence TF-1 cells) 37101A60R, V161A M1 1.0 1394867 I63A, V67T, V161N, P162A M2 1.0 12394867I63T, V67A, V161N, P162A M3 1.5 1374972 M55A, I63A, V67A, V161A M4 0.21374973 M55T, I63A, V67A, V161A M5 0.1 12484973 M55T, I63T, V67A, V161AM6 0.1 1374968 Q61A, I63A, V67A, V161A M7 0.4 12484968 Q61A, I63T, V67A,V161T M8 1.4 124849 I63T, V67A, V161T M9 0.4 123749 I63T, V67A, V161AM10 1.0 13749 I63A, V67A, V161A M11 0.5 14849 I63A, V67A, V161T M12 1.0124860 I63T, V67T, V161T M13 1.0 3849 V67A, V161R M14 0.6 4849 V67A,V161T M15 0.4 3760 V67T, V161A M16 0.7 4860 V67T, V161T M17 0.9 3749V67A, V161A M18 0.2 4249 V67A, V161E M19 1.0 1248 I63T, V161T M20 0.71238 I63T, V161R M21 1.8 1242 I63T, V161E M22 0.4 1237 I63T, V161A M231.5 149 I63A, V67A M24 1.5 160 I63A, V67T M25 2.0 1249 I63T, V67A M261.6 137 I63A, V161A M27 3.0 142 I63A, V161E M28 1.5 138 I63A, V161R M293.3 148 I63A, V161T M30 4.0 6063 E50A, V67T M31 1.0 163 E50A, I63A M324.8 1263 E50A, I63T M33 6.3 4263 E50A, V161E M34 5.0 37 V161A M35 1.0 40V161D M36 1.0 42 V161E M37 0.5 43 V161G M38 1.0 44 V161H M39 1.0 39V161N M40 1.0 46 V161P M41 1.0 45 V161K M42 3.3 41 V161Q M43 1.0 38V161R M44 2.0 48 V161T M45 1.0 49 V67A M46 0.5 60 V67T M47 0.6 1 I63AM48 2.0 12 I63T M49 2.0 68 Q61A M50 0.6 69 Q61T M51 2.3 72 M55A M52 0.3102 M55K M53 0.1 74 M55S M54 1.0 73 M55T M55 0.1 100 T58A M56 1.0 35W51S M57 8.0 63 E50A M58 1.0 77 L69A M59 1.0 78 L69S M60 0.7 79 L69T M610.2 83 L71A M62 1.8 86 E72A M63 1.8 87 E72S M64 2.1 101 A60R M65 1.4101102 M55K, A60R M66 0.2 37101102 M55K, A60R, V161A M67 0.4 00 WT M68 —*Relative activity is ED₅₀ test protein (M1–M67)/ED₅₀ Fc-TPO WT (M68).ED₅₀ measured in TF-1 cells.

Example 5

Identification of T-Cell Epitopes in Human TPO

All blood samples used in this study were obtained with approval of theAddenbrooke's Hospital Local Research Ethics Committee. T-cell epitopemapping was performed using human PBMCs isolated from blood obtainedfrom the National Blood Transfusion Service (Addenbrooke's Hospital,Cambridge, UK). PBMCs from 20 healthy donors were isolated by Ficolldensity centrifugation and stored under liquid nitrogen. Each donor wastissue-typed using an Allset™ PCR based tissue-typing kit (Dynal) and Tcell assays were performed by selecting donors according to individualMHC haplotypes. 15 mer peptides staggered by three amino acids andspanning the human TPO sequence between residues 1-177 were purchasedfrom Pepscan Systems BV (NL). Using this scheme, total of 55 peptideswere required to scan the TPO residues of interest. The sequence andpeptide number of these peptides are provided in Table 3.

TABLE 3 Peptides used to map immunogenic epitopes within TPO (Peptidesspan TPO residues 1-177) Peptide SEQ ID No Peptide sequence NO: 1SPAPPACDLRVLSKL 74 2 PPACDLRVLSKLLRD 75 3 CDLRVLSKLLRDSHV 76 4RVLSKLLRDSHVLHS 77 5 SKLLRDSHVLHSRLS 78 6 LRDSHVLHSRLSQCP 79 7SHVLHSRLSQCPEVH 80 8 LHSRLSQCPEVHPLP 81 9 RLSQCPEVHPLPTPV 82 10QCPEVHPLPTPVLLP 83 11 EVHPLPTPVLLPAVD 84 12 PLPTPVLLPAVDFSL 85 13TPVLLPAVDFSLGEW 86 14 LLPAVDFSLGEWKTQ 87 15 AVDFSLGEWKTQMEE 88 16FSLGEWKTQMEETKA 89 17 GEWKTQMEETKAQDI 90 18 KTQMEETKAQDILGA 91 19MEETKAQDILGAVTL 92 20 TKAQDILGAVTLLLE 93 21 QDILGAVTLLLEGVM 94 22LGAVTLLLEGVMAAR 95 23 VTLLLEGVMAARGQL 96 24 LLEGVMAARGQLGPT 97 25GVMAARGQLGPTCLS 98 26 AARGQLGPTCLSSLL 99 27 GQLGPTCLSSLLGQL 100 28GPTCLSSLLGQLSGQ 101 29 CLSSLLGQLSGQVRL 102 30 SLLGQLSGQVRLLLG 103 31GQLSGQVRLLLGALQ 104 32 SGQVRLLLGALQSLL 105 33 VRLLLGALQSLLGTQ 106 34LLGALQSLLGTQLPP 107 35 ALQSLLGTQLPPQGR 108 36 SLLGTQLPPQGRTTA 109 37GTQLPPQGRTTAHKD 110 38 LPPQGRTTAHKDPNA 111 39 QGRTTAHKDPNAIFL 112 40TTAHKDPNAIFLSFQ 113 41 HKDPNAIFLSFQHLL 114 42 PNAIFLSFQHLLRGK 115 43IFLSFQHLLRGKVRF 116 44 SFQHLLRGKVRFLML 117 45 HLLRGKVRFLMLVGG 118 46RGKVRFLMLVGGSTL 119 47 VRFLMLVGGSTLCVR 120 48 LMLVGGSTLCVRRAP 121 49VGGSTLCVRRAPPTT 122 50 STLCVRRAPPTTAVP 123 51 CVRRAPPTTAVPSRT 124 52RAPPTTAVPSRTSLV 125 53 PTTAVPSRTSLVLTL 126 54 AVPSRTSLVLTLNEL 127 55SRTSLVLTLNELPNR 128

For each donor sample, PBMCs were thawed and resuspended in AIM-V(Invitrogen) containing 100 units/ml penicillin, 100 ug/ml streptomycinand 1 mM glutamine. Triplicate cultures of 2×10⁵ PBMC/well offlat-bottomed 96 well plate were incubated with peptides at a finalconcentration of 1 μM and 10 μM. Cells were incubated for 7 days beforepulsing with 1 μCi/well tritiated thymidine for 18 hours. Cultures wereharvested onto glass fibre filter mats using a Tomtec Mach III plateharvester and cpm values determined by scintillation counting using aWallac Microbeta TriLux plate reader.

The results of these assays are depicted in FIG. 1. Regions ofimmunogenicity (FIG. 1A) were determined by identifying peptides thatinduced donors to respond with stimulation indexes≧2 and bydetermination of the donor response rate for each peptide. Peptideslocated within two separate regions were able to induce T cellproliferation. Region 1 encompasses TPO residues 49-75 and comprises thesequence: GEWKTQMEETKAQDILGAVTLLLEGVM (SEQ ID NO: 2) and equivalent topeptides 17-21. The donor responses to region 1 ranged from 13% to 17%.Region 2 encompasses TPO residues 157-171 and comprises the sequence:PTTAVPSRTSLVLTL (SEQ ID NO: 3) (peptide 53). The donor response rate toregion 2 was 13% (FIG. 1B). Each donor was also tested for their abilityto respond to two positive control peptides influenza haemagglutinin Aamino acids 307-319 [J. I. Krieger et al. (1991) Journal of Immunology;146: 2331-2340] and chlamydia HSP60 amino acids 125-140 [M. C. Cerroneet al. (1991) Infection and Immunity; 59: 79-90]. Keyhole limpethaemocyanin, a well documented potent T cell antigen was also used as acontrol.

Example 6

Analysis of Immunogenic Regions by Time-Course T-Cell Assays

Bulk cultures of 2-4×10⁶ PBMC/well were established from 20 healthydonor samples in 24 well plates. Cells were incubated for 6 to 9 dayswith WT and variant peptides spanning the immunogenic regions (see Table4). T cell proliferation was assessed by tritiated thymidineincorporation on days 6, 7, 8 and 9. Proliferation was assessed at eachtime point, by gently resuspending the bulk cultures and removingsamples of PBMC, that were then incubated in triplicate wells ofU-bottomed 96 well plate with 1 μCi/well tritiated thymidine for 18hours as described above.

The time course assay was used to test variant peptides containingsubstitutions over WT. Substitutions were made at key locations wherethere was expectation that the substitution would prevent binding to MHCclass II and therefore, subsequent T cell proliferation in the assay.Particular substitutions were made based on information from the crystalstructure of the cytokine domain of TPO [Feese M. D. et al (2004) Proc.Natl. Acad. Sci (USA); 101: 1816-1821] and various models of MHC classII binding motifs. The favoured mutations were large basic residues suchas arginine or lysine but where structural models predicted severeaffects on the protein structure we used alanine instead. For Region 2we used four different alternatives for one locus as we only identifiedone important residue for mutagenesis studies in that sequence.

Peptides containing the mutations: M55K, T58R A60R, D62R, L69A, L70Awere synthesised for region 1, and V161A, V161N, V161R and V161T forregion 2 (Table 4).

Results of this analysis are shown in FIGS. 2 and 3. FIGS. 2A and 3Ashow that 15% of the 20 donors responded to the original sequence region1 sequence and T58R. By contrast none of the donors responded to themutated sequences containing the M55K or the A60R mutations. FIGS. 2Band 3B show that 25% of the 20 donors responded to the original region 2sequence and V161T. By contrast none of the donors responded to themutated sequences containing the V161A or V161R mutations.

TABLE 4 Sequences of peptides used in time-course assays ImmunogenicRegion Wild Type Sequence Modified Sequences R1GEWKTQMEETKAQDILGAVTLLLEGVM GEWKTQKEETKAQDILGAVTLLLEGVM (SEQ ID NO: 129)GEWKTQMEERKAQDILGAVTLLLEGVM (SEQ ID NO: 130) GEWKTQMEETKRQDILGAVTLLLEGVM(SEQ ID NO: 131) GEWKTQMEETKAQRILGAVTLLLEGVM (SEQ ID NO: 132)GEWKTQMEETKAQDILGAVTALLEGVM (SEQ ID NO: 133) GEWKTQMEETKAQDILGAVTLALEGVM(SEQ ID NO: 134) R2 PTTAVPSRTSLVLTL PTTAAPSRTSLVLTL (SEQ ID NO: 135)PTTANPSRTSLVLTL (SEQ ID NO: 136) PTTARPSRTSLVLTL (SEQ ID NO: 137)PTTATPSRTSLVLTL (SEQ ID NO: 138)

Example 7

Functional Activity of Most Preferred TPO Muteins

Proliferative activity variants of non-immunogenic for both epitoperegion 1 and epitope region 2 were tested. Supernatants were quantifiedby Fc ELISA and diluted to 160 ng/ml. The protein activity was titratedin 2 fold serial dilutions and tested in the TF-1 cell proliferationassay. Those changes that gave ≧100% of wild-type activity, andabrogated the T cell proliferative response for both region 1 and region2, are the most preferred embodiments of the present invention. Relativeactivity values for all functional muteins are listed in Table 2.

A further preferred TPO mutein is clone ID 102 # (M52/F1-L1-M52)comprising substitution M55K. The M55K substitution was surprisinglyshown to produce a beneficial increase in potency of the molecule. Thiseffect is demonstrated in FIG. 4 where the activity in the TF-1proliferation assay is plotted versus protein concentration. Muteinclone ID # 102 shows significantly greater activity in this functionalassay than either the WT counterpart or a control TPO preparationlacking the Fc domain.

Table 5 provides a listing to the relative activites of the mostpreferred muteins of the invention. The relative activites are derivedby dividing the ED₅₀ values scored for the test protein by that of theFc-gamma 4-linked WT TPO (M68/F1-L1-M68). A relative acitivity of 1.0 orless represents a protein with equal or better activity than WT. Valuesgreater than 1.0 indicate inferior activity. The relative activity ofthe full length WT recombinant human TPO (r-hTPO) is also provided. Allvalues are the average of at least three separate determinations.

TABLE 5 Relative acitivity of most preferred TPO muteins F1-L1-M CloneID Sequence No Substitution set Relative Activity 37101 M1 A60R, V161A 1101102 M66 M55K, A60R 0.2 37101102 M67 M55k, A60R, V161A 0.4 102 M53M55K 0.1 r-hTPO N/a WT 2.5

A further example of an especially preferred molecule of the inventionwith significantly enhanced activity provided by the TPO muteincontaining the substitiution set M55K, A60R, V161A (M67/F1-L1-M67). Thisprotein is highly potent in the TF-1 assay with relative activity ofaround 0.4.

Although the M55K, A60R, V161A (M67/F1-L1-M67) mutein is clearly ahighly potent molecule, this mutein is not as active as the muteincomprising only the M55K and A60R substitutions (M66/F1-L1-M66). Thismutein demonstrates a relative activity of 0.2.

Some of the muteins with two or more substitutions were then tested inthe TF-1 and M0-7e proliferation assays. A most preferred protein of theinvention Fc-gamma 4-L-TPO (A60R V161A) (M1/F1-L1-M1) was found toretain full activity. The activity of this molecule was compared withboth an Fc-gamma 4 linked TPO 1-174 WT molecule (M68/F1-L1-M68), and apreparation of full-length recombinant human (r-hTPO) (PeproTech,London, UK). Results are shown in Table 6.

In TF-1 cells, the native fusion protein, has an ED₅₀ of 12.0 ng/ml. InM0-7e cells, the native fusion protein, has an ED₅₀ value of 25.0 ng/ml.By contrast, it has been somewhat surprisingly found that a mostpreferred molecule of the invention (M1/F1-L1-M1) has an ED₅₀ value inTF-1 cells of 11.5 ng/ml and in M0-7e cells an ED₅₀ value of 18.0 ng/ml.Given that this molecule is a TPO mutein, these results indicate thatthe changes to the sequence have had a beneficial effect on directfunctional activity.

Enhanced potency in the molecule attributed to the dimeric nature of theprotein by virtue of the Fc-domain is demonstrated by comparison of theED₅₀ values found using the full size (non-Fc linked) human TPO moleculein TF-1 and E0-7e based proliferation assays (Table 6). Monomericrecombinant human (r-hTPO) TPO achieves an ED₅₀ of 29.5 ng/ml using TF-1cells and 70.0 ng/ml using M0-7e cells. A most preferred molecule of theinvention therefore demonstrates approximately between 2.5-4.0 foldenhanced activity over r-hTPO.

Table 6 provides a comparison of the ED50 values scored in both TF-1 andM0-e7 cells; Values shown are for the Fc-gamma 4-linked TPO mutein A60RV161A (M1/F1-L1-M1), the WT Fc-linked counterpart (M68/F1-L1-M68) and afull length WT recombinant human TPO (r-hTPO). Each value is the averageof three separate measurements.

TABLE 6 Activities of Fc-gamma 4-linked TPO mutein A60R V161A(M1/F1-L1-M1) with WT TPO Fc-linked counterpart proteins. ProliferationActivity Assay ED₅₀ ng/ml Test Protein TF-1 cells M0−7e cells Clone ID37101 - A60R, V161A 11.5 18.0 (M1/F1-L1-M1) Clone ID 00 - WT 12.0 25.0(M68/F1-L1-M68) r-hTPO 29.5 70.0

1. An isolated polypeptide comprising the amino acid residue sequence ofSEQ ID NO:
 6. 2. A fusion protein comprising a polypeptide of claim 1linked at the N-terminus thereof to a human immunoglobulin Fc regionpeptide.
 3. The fusion protein of claim 2 wherein the Fc region peptideis linked to the N-terminus of the polypeptide by a linking peptideconsisting of the amino acid residue sequence of SEQ ID NO:
 5. 4. Thefusion protein of claim 2 wherein the Fc region peptide is a human IgG4Fc region peptide.
 5. The fusion protein of claim 4 wherein the humanIgG4 Fc region peptide is linked to the N-terminus of the polypeptide bya linking peptide consisting of the amino acid residue sequence of SEQID NO:
 5. 6. An isolated peptide molecule consisting of SEQ ID NO: 6.